Receptor for natural killer and non-specific cytotoxic cells

ABSTRACT

The present invention encompasses a receptor protein present on the surface of natural killer cells (NK cells) and non-specific cytotoxic cells (NCC) which is involved in non-specific lysis of target cells bearing an antigen recognized by the receptor protein and monoclonal antibodies which bind to the receptor which are useful in their identification and purification, and methods for altering NK cell-mediated lysis of target cells. 
     The monoclonal antibodies (mAbs) of the present invention were prepared by cell fusions between spleen cells from mice immunized with either non-specific cytotoxic cells (NCC) from catfish (anti-receptor antibodies) and myeloma cells. 
     The NK cell receptor was purified from solubilized flow cytometry purified non-specific cytotoxic (NCC) using antigen-antibody complexing techniques, either immune precipitation or affinity chromatography on Affigel-10™ with Con A Sepharose purified mAbs. Using mAbs which bind but do not inhibit lysis produces the same results as using mAbs which bind and inhibit lysis. Analysis of the receptor by 12% SDS shows that it is a dimeric molecule consisting of 41,000 and 38,000 D proteins.

The U.S. government has certain rights in this invention by virtue ofgrants from the U. S. Department of Agriculture.

This is a continuation of U.S. Ser. No. 07/109,772 filed Oct. 16, 1987now abandoned.

This invention is generally in the area of immunology and proteincharacterization and specifically in the area of protein receptors onnatural killer cells.

BACKGROUND OF THE INVENTION

By the early 1970's, natural killer (NK) cell activity, distinct fromantigen specific cytolytic T lymphocyte activity, had been reported byseveral laboratories in a variety of species, including mouse, rat andman. These reports are reviewed by R. K. Oldham, J. Biol. Resp. Mod. 1,217 (1982). Non-specific cytotoxic cells (NCC), similar in many aspectsto NK cells, also have been identified in lower vertebrate species(Graves et al., Dev. Comp. Immunol. 8, 293 (1984); Evans et al., Dev.Comp. Immunol. 8, 303 (1984); Evans et al., Dev. Comp. Immuno. 8, 599(1984); Evans et al., Dev. Comp. Immunol. 8, 823 (1984)).

NK cells are known to be a heterogeneous population of immune cells withregard to phenotype and function, as discussed, for example, by Lanieret al., Immunol. Today, 7, 132 (1986). However, controversy still existswith regard to their lineage with respect to other immune cells and totheir relationship to other effector cells such as lymphokine-activatedkiller (LAK) cells and anomalous killer (AK) cells. These differencesmay reflect the innate heterogeneity of the types of cells capable of NKactivity (as defined by function) or be due to the various assay systemsemployed to study NK cells.

NK cells have been implicated in a variety of activities involving theimmune system, including immune disorders in both animal models and man.Results from animal models have also shown that NK cells are effectivein vivo against the growth and metastasis of certain types of tumors. Inthis regard, administration of in vitro cultured NK cells to humancancer patients has shown promise in the treatment and regression ofcertain types of malignancies. Further, NK cells have been implicated inhost resistance to infections with microorganisms, both bacteria andviruses. Finally, NK cells are thought to play an important role in thenormal regulation of the host immune system through immunoglobulinproduction and hematopoiesis. These lines of evidence suggest that theNK system possesses considerable functional diversity, and may operateby the recognition of changes in normal membrane structures, includingdifferentiation antigens, as discussed by Toshitani et al., CellImmunol. 108, 188 (1987); Kornbluth et al., J. Immunol. 134, 728 (1985);Lauzon and Roder, Cell Immunol. 94, 85 (1985); Roder et al., J. Exp.Med. 150, 471 (1979).

Although NK cells have been studied for several decades, two of themajor questions that remain in NK biology today are what molecule(s) onthe surface of NK cells is involved in recognition of the target cellsand what molecule(s) on the target cells is recognized by NK cells. Atthe beginning of the last decade it was found that T cells recognizeproducts of the major histocompatibility complex (MHC) expressed ontarget cells. This discovery was facilitated by the availability ofmonoclonal antibodies (mAbs) against these molecules which inhibitedtheir recognition by T cells. In terms of NK biology, mAb inhibition offunction or recognition (in the absence of complement) has been muchless frequently documented. Both the transferrin receptor and thereceptor for IgG Fc have been implicated to serve as recognitionstructures for NK cells, although these results are controversial(Vodinelich et al., Proc. Natl. Acad. Sci. USA 80, 835 (1983); Dokhelaret al., Eur. J. Immunol. 14, 340 (1984). The laminin/laminin receptorcomplex has also recently been implicated to act as a means of NKrecognition of certain cells, as reported at the Fourth Int'l Workshopon NK Cells, Kingston, Ontario, 1986. Finally, carbohydrate/carbohydrateinteractions have been implicated to serve as a means of NK/target cellrecognition by Muchmore et al., Immunolbiol. 158, 191 (1981);Werkmeister et al., Cell Immunol. 80, 172 (1983). Together, theseresults have been interpreted as signifying that NK cells do not expressclonally-restricted receptors, that NK cells may express more than onereceptor on their surface and that NK cells are capable of recognizingmultiple antigens on the surface of one or more target cells. Theseresults may also be indicative of NK heterogeneity due to discrete NKsubpopulations.

Some typical T cells, particularly antigen-specific cytotoxic T cells(CTL), can mediate NK-like cytotoxicity as measured against certaintumor cells such as K562, as described by Brooks et al., Immunol. Rev.72, 43 (1983) and others. These CTLs are referred to as CTL(NK) ornon-MHC-restricted CTL. However, since it has been shown that true NKcells do not transcribe mRNA for or express on their surface a typical Tcell antigen receptor (TCR) (Reynolds et al., J. Exp. Med. 150, 471(1985); Lanier et al., J. Exp. Med. 163, 209 (1986); Tutt et al., J.Immunol. 137, 2998 (1986)), it is generally accepted that NK cells donot recognize antigen in this fashion and do not recognize MHCmolecules. Although the CTL(NK) do express a typical TCR, it is notknown whether this molecule is utilized to effect their NK-like killingor if these effector cells recognize MHC antigens in some aberrantfashion. Recently, there has been evidence to suggest that the CD3molecule and the gamma/delta TCR may be involved in this type ofnon-MHC-restricted cytotoxicity (Farrini et al., J. Exp. Med. 166, 277(1987); Alarcon et al., Proc. Natl. Acad. Sci. USA 84, 3861 (1987); Anget al., J. Exp. Med. 165, 1453 (1987); David et al., J. Immunol. 138,2831 (1987); van de Grien et al., J. Immunol. 138,1627 (1987)). In otherstudies, it has been demonstrated that anti-CD3 and anti-CD8 mAbs, whileinhibiting the antigen-specific lysis by such CTL clones, do not inhibitthe NK-like lysis by these effector cells (Moretta et al., Eur. J.Immunol. 14, 121 (1984); Brooks and Holscher, J. Immunol. 6, 594 (1987);Roberts and Moore, Eur. J. Immunol. 15,448 (1985)).

                  TABLE 1                                                         ______________________________________                                        Natural killer cell membrane antigens and mAbs capable                        of detecting these determinants.                                              mAb    Designation                                                                             Antigen Specificity                                          ______________________________________                                        CD2    (Leu-5b)  T cells, NK cells                                            CD7    (Leu-9)   T cells, NK cells                                            CD8    (Leu-2)   T cells, NK cells                                            CD11   (Leu-15)  C3bi receptor on T Cells, NK cells,                                           momonocytes and macrophages                                  CD16   (Leu-11a) Fc (gamma) receptor on NK cells and                                           on PMN's                                                     Leu 7            HNK-1 on LGL's and NK cells                                  Leu 19           NKH-1 determinant (220 kD) on NK and                                          T-cells                                                      .sup.a) OKT8     T cytotoxic/suppressor cells and LGL                         OKT10            LGL, early thymus antigen                                    3A1              T cells and LGL                                              5A12             T cells and LGL                                              Lyt 3            T cells and LGL                                              OKT5             T cytotoxic/suppressors and LGL                              Ia               Activated T cells                                            ______________________________________                                         .sup.a) Cells expressing OKT8, OKT5 and Ia are nonlytic LGL's.           

Very little is known regarding identification of lymphoreticular cellsin fish. Previous studies have tentatively identified a B lymphocytesubpopulation in this species. However, there have been only a fewinvestigations where attempts have been made to identify cytotoxic cellsin teleost fish. In mammals, however, a great deal is known regardingthe heterogeneity of lymphocytes and mAbs have been derived whichinhibit cytotoxic activity.

It therefore seems apparent that in order to understand the process ofNK cell recognition, it is essential to identify and characterizeexamples of NK target antigens, as well as the receptor(s), in a varietyof species, including lower vertebrates such as fish and mammals.

Knowledge of an NK antigen receptor would enable analysis of how thereceptor/ligand complex functions. Further, isolation of an NK antigenreceptor would allow comparison with other receptors and possibly theidentification of other NK antigen receptors or subclasses of NK cellson the basis of antigen specificity and/or type of antigen receptor.This would further the understanding of the relationship between NK, LAKand CTL(NK) populations as well as immune cells such as monocytes,macrophages and other types of T cells. A comparison of the receptormolecules in fish, mouse and humans provides insights into the evolutionof the NK cell population and the role they play in immune function,malignancy and immune disorders.

It is therefore an object of the present invention to provide methodsand means for isolating and characterizing the antigen receptor commonto the surface of natural killer cells of such diverse origin as fish,mouse and human.

It is a still further object of the present invention to provide methodsand means for modifying the antigen receptor of natural killer cells andthe immune response modulated by the receptor.

SUMMARY OF THE INVENTION

The present invention encompasses a receptor molecule (dimer) present onthe surface of natural killer cells (NK cells) and non-specificcytotoxic cells (NCC) which is involved in non-specific lysis of targetcells bearing an antigen recognized by the receptor and methods foraltering NK cell-mediated lysis of target cells.

The anti-receptor monoclonal antibodies (mAbs) of the present inventionwere prepared by cell fusions between spleen cells from mice immunizedwith either non-specific cytotoxic cells (NCC) from catfish and myelomacells. Four distinct mAbs have been characterized against the NK/NCCcell receptor. As used in the present invention "NK cells" will be usedto include both natural killer cells of mammalian origin andnon-specific cytotoxic cells of fish origin, unless specifically statedotherwise. Two of the mAbs directed against the receptor are able tosignificantly inhibit NK lysis at low mAb concentrations, as assessed byinhibition of cellular cytotoxicity responses. Two of the mAbs directedagainst the receptor bind to the protein(s) but do not inhibit lysis.

The NK cell receptor was purified from solubilized flow cytometrypurified non-specific cytotoxic cells (NCC) using antigen-antibodycomplexing techniques, either immune precipitation or affinitychromatography on Affigel-10™ with Con-A Sepharose purified mAbs. UsingmAbs which bind but do not inhibit lysis produces the same results asusing mAbs which bind and inhibit lysis. Analysis of the receptor by 12%SDS polyacrylamide gel electrophoresis (PAGE) shows that it is a dimericmolecule consisting of subunits of approximately 41,000 and 38,000Daltons, respectively. The exact chemical structure of the NK receptorwhich is recognized by the mAbs is presently unknown. Although probablyconsisting of glycoproteins, this dimer may be composed of complexpolysaccharides, lipids, or lipoproteins, mucopolysaccharides or acombination of these different structures. The molecule is furthercharacterized by radiolabeling surface protein and carbohydrate, gelelectrophoresis, isoelectricfocusing, and peptide mapping. Pulselabeling with 35S-methionine or 3H-leucine/3H-lysine can be used todetermine the rate of turnover of the molecule. In combination withtunicamycin treatment, the extent and importance of glycosylation can bedetermined. Treatment with neuraminidase, endoglycosidase-F andendoglycosidase-H can be used to study processing of the matureglycoprotein.

The gene sequence for the receptor can be identified and probes for theprotein isolated by constructing a cDNA library containing mRNA codingfor the receptor. By sequencing a portion of the purified protein, a17-20 base oligonucleotide mixture can be used to screen cDNA librariesprepared from mRNA from NCC inserted into an expression vector such aslambda gt11. Similarly, antibodies to the receptor can be used to screenthe proteins expressed from the vector libraries and the positive clonessequenced and used as further probes. The entire sequence can bedetermined by sequencing overlapping deletion fragments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 compares the inhibitory effect of mAbs 5C6.10.4 and 6D3.4.4 onthe lysis by NCC of ⁵¹ Cr-labeled NC-37 target cells. Effector cellswere incubated with 10 μg of mAb for 4 h, washed 2x, and mixed withtarget cells at an effector to target cell ratio of 20:1, 40:1, 80:1, or160:1.

FIG. 2 compares the inhibitory effect of 10 μg 5C6.10.4 and 2B2.4.9 mAbdirected against NCC on lysis of anterior kidney cells by NCC in a 4 hassay at 27° C., where sorted effector cells are enriched by flowcytometry at effector to target cell ratios of 20:1, 40:1, 80:1, and160:1.

FIG. 3 compares the percent lysis of ⁵¹ Cr-labelled K562 targets (10,000targets/well) incubated with endogenous NK or activated NK cells. Cellswere either preincubated with media as a control or with 2, 5, or 10 μgmAb/well for 1 h at 4° C., in a 3 h assay at 37° C., at effector totarget cell ratios of 1:1, 3:1, and 10:1.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a receptor protein found on the surface ofnatural killer cells and non-specific cytotoxic cells (jointly referredto hereafter as "NK cells" unless stated otherwise) in a variety ofspecies, including man, mouse, rat, and fish that mediates lysis of theantigen bearing target cells. The present invention also includesmethods for receptor isolation, characterization and use under a varietyof conditions.

Non-specific cytotoxic cells (NCC) represent a population of cells thatmediate "natural" cellular immunity in the catfish (I. punctatus),analogous to mammalian natural killer (NK) cells. NCC bind to and lyse avariety of transformed murine and human B-cell, T-cell and myeloidtargets. Similar to NK cells, NCC require cell-cell contact forinitiation of the first stage of lysis; NCC are plastic and nylon woolnonadherent; NCC require a period of preincubation prior to addition oftarget cells to augment killing; and, like NK cells, NCC have similarMg⁺⁺ and Ca⁺⁺ requirements for binding and killing, respectively.

Additional comparisons have been made between NCC and NK cells usingMichaelis-Menten kinetics and Lineweaver-Burk transformation studies.Approximately five times more NCC cells are required to kill anindividual target cell compared to NK cells and, in a three hour killingassay, NCC do not recycle. However, NCC may be more pluripotent andfunctionally undifferentiated when compared to mammalian NK cells, asshown by comparisons demonstrating rapid killing kinetics, extremelywide temperature optima and multiple target cell phenotypespecificities. NCC produce rapid lysis of target cells (90% of totallysis within 90 minutes) and NCC bind to and lyse a wide variety ofdifferent types of target cells. These targets included YAC-1, P815,NC-37, DAUDI, P3HR-1, MOLT-4, K562, U937, and HL-60 cells.

This very large spectrum of target cell killing indicates either thatrecognition is entirely antigen non-specific or that two differentdeterminants are required for NCC recognition and lysis of a target.Cold target inhibition has shown that NCC are comprised of manydifferent subpopulations of antigen specific cells and that homologous,but not heterologous cold targets, inhibit lysis. In addition, cellswhich have similar properties, such as certain Epstein-Barr transformedB-cells (NC-37, P3HR-1, DAUDI) which are susceptible to lysis, canreciprocally act as cold targets to inhibit lysis by NCC. These datasuggested that multiple different target cells could be recognized bydifferent subsets of NCC, and that clonotypic-like NCC functions couldbe mediated by a specific antigen receptor(s).

Isolation of mAbs Directed Against a Target Cell Antigen. Since NCClysis is characterized as being non-MHC-restricted and NCC are capableof lysing a variety of transformed human cell lines, including NC-37 (ahuman lymphoblastoid B-cell line), DAUDI (a human lymphoblastic B-cellline) and P3HR-1 (Same as DAUDI), and the naturally occurring fishparasite Tetrahymena pyriformis as well as several murine cell linessuch as YAC-1 (a mouse lymphoma induced by Moloney Leukemia virus), andbecause the parasite targets will cold-compete with the tumor cell linesin cytotoxicity assays, it appeared that certain human and murinetransformed cell lines shared some common antigenic determinant with afish parasite that is recognized by fish NCC. For this reason, the NC-37cell line was used as a source of antigen in the generation of mAbs.

Spleen cells from NC-37 primed Balb/c mice were fused with P3-X63-Ag8.653 myeloma cells. The resulting mAbs were screened with Tetrahymenapyriformis and the NC-37 cell line for those mAbs which reacted withboth in an ELISA. Four independent positive binding mAbs then werechosen for expansion and cloning by repeated limiting dilution. ThesemAbs are designated 1E7, 18C2.8.3, ID4 and 7C6.5.4. 18C2.8.3, whichproduces IgM mAbs that bind to target antigen and inhibit NK-mediatedlysis, and 7C6.5.4, which produces IgG mAbs that bind to target antigenbut do not inhibit NK-mediated lysis, were deposited with the AmericanType Culture Collection, Rockville, Md., on Oct. 16, 1987 and designatedATCC numbers HB9571 and HB9574, respectively. All of the mAbs were grownin ascites and purified from the ascites by Sepharose-Protein A (IgG) orSepharose-Con A (IgM) chromatography prior to testing.

Characterization of mAb Directed Against A Target Cell Antigen. Thefollowing studies show that monoclonal antibodies derived against NC-37cells specifically inhibit fish NCC lysis of target cells. Inhibition ofcytotoxicity by mAbs 18C2.8.3 and 1E7 is dose dependent with inhibitionlocated at the target cell level. All of the target cell lines studied,whether of human or of mouse origin, are protected from NCC lysis byprior treatment with these mAbs. Other mAbs (7C6.5.4 and 1D4) bind tothe target cells but do not inhibit lysis. These data indicate that thedeterminants recognized by mAbs 18C2.8.3 and 1E7 are the same (or atleast part of the same antigenic complex) and that inhibition of lysisis probably not the result of steric hindrance.

The widespread cellular distribution of these mAb-defined antigenicdeterminants was further shown by experiments demonstrating inhibitionof lysis of Tetrahymena pyriformis by mAb 1E7. In addition, adsorptionof the inhibitory mAb with different numbers of the parasite selectivelyremoved the inhibitory activity of the mAb when tested in a lytic assayagainst NC-37 target cells. This result demonstrated that the mAbrecognized the same antigenic determinant(s) on both mammalian andprotozoan target cells.

Isolation and Characterization of a Target Cell Antigen. Biochemicalanalysis of the antigens recognized by the target cell specific mAbs wasaccomplished by Western blotting and by immunoprecipitation studies.Both techniques revealed that each inhibitory mAb bound to two proteins.Using Western blot analysis, one molecule was of 42,000 D while thesecond was either of 78,000 D or 86,000 D depending on which mAb wasused (mAbs 1E7 or 18C2.8.3, respectively). Similar to the Western blotanalysis, immunoprecipitates with either mAb 1E7 or 18C2.8.3 revealed a42,000 D antigen. Unlike Western blot analysis, however, the second andlarger molecule precipitated was of 80,000 D. The apparent differencesin molecular weight exhibited by the 78, 80, and 86 kD molecules maypotentially be due to variation in glycosylation or gel-to-gel variationduring SDS-PAGE analysis. It is possible that the larger protein, ofapproximately 80 kD, may be a dimer of the 42,000 D antigen. Usingdifferent reducing conditions (varying β-mercaptoethanol concentrationsand times/temperatures of incubation) did not produce a single band at42,000 D.

The results indicate that the target cell antigen recognized by thesemAbs is involved in the recognition process necessary for lysis oftarget cells by fish NCC and has important implications in the study ofhost defense, tumor biology, and the recognition process of cytotoxiccells.

³⁵ S-methionine-labeled NC-37 cell lysates were precipitated with mAbs18C2.8.3 and 1E7 bound to protein A beads, eluted, and separated byelectrophoresis on 11% SDS-PAGE. Two major antigens were eluted from theimmune complexes with molecular weights of 42,000 D and 80,000 D,respectively.

These observations are in agreement with the primary molecule observedin the immunoprecipitation experiments with these mAbs and NC-37 tumorcells, which appears to be a dimeric complex. Based on the binding data,the mAbs do not appear to recognize Fc receptors, transferrin receptors,laminin receptors or laminin, immunoglobulin, Epstein-Barr viralantigens or MHC antigens. In fact, antibodies directed against MHCmolecules, which are very conserved in structure, do not recognize suchmolecules across such disparate species. Thus, it appears that the mAbsdefine novel, simple and conserved molecules which serve as NK targetantigens in the process of NK lysis. This molecule probably evolvedearly in the evolution of the immune system to function in regulation ofthe developing immune system. In fact, this molecule may have some asyet undetermined physiological role.

Isolation and Characterization of mAbs Directed Against NK CellReceptor. mAbs were produced against the NK cell receptor using asimilar methodology to that described with respect to the antigen. mAbswere derived against flow cytometry purified fish (Ictalurus punctatus)non-specific cytotoxic cells (NCC). Four mAbs were cloned andcharacterized. mAbs 5C6.10.4 and 6D3.2.10 produce 60-65% inhibition oflysis of NC-37 target cells (a human B-lymphoblastoid cell line) byunfractionated NCC. MAbs 2B2.4.9 and 6D3.4.4 are non-inhibitors ofcytotoxicity. All mAbs are the same isotype (IgM) and are cloned bylimiting dilution (2×). Inhibitory activity is specific for the effectorcells because the mAbs have no effect on NCC cytotoxicity when only thetarget cells are treated. Inhibition can be produced by preincubation ofthe mAbs with NCC or by no preincubation. Inhibition is not reversible.Approximately 100% inhibition is produced by treatment of flow cytometry(FCM) purified NCC with the inhibitor mAbs. Inhibition of cytolysis wasspecific for the effector cells and not the target cells sincepreincubation of the target cells with mAbs 5C6.10.4 (inhibitory mAb) or6D3.4.4 (noninhibitory mAb) followed by removal of the mAbs prior to theaddition of the effector cells did not inhibit lysis. Both inhibitormAbs significantly inhibit conjugate formation between effector andNC-37 target cells.

Two of the mAb directed against the NK cell receptor, 5C6.10.4 and6D3.4.4 were deposited with the American Type Culture Collection on Oct.16, 1987 and designated HB9572 and HB9573, respectively.

Flow Cytometry Characterization of mAbs Directed Against the NK cellReceptor. Flow cytometry is also used to determine mAb binding tovarious effector cell populations. Inhibitor and non-inhibitor mAbs bindto approximately 25% (5C6.10.4) and 39% (6D3 4.4) of fish anteriorkidney cells; to 42% (5C6.10.4) and 54% (6D3.4.4) of fish spleen cells;and to 2.5% (5C6.10.4 and 6D3.4.4) of fish peripheral blood.

To determine the requirements needed to produce optimal inhibition ofNCC lysis, each mAb was first purified using Con-A Sepharose. The mAbswere next either preincubated with the effector cells for 4 hours (andwashed 2×) or not preincubated but added at the start of thecytotoxicity assay. Both incubation protocols were equally efficient ininhibiting NCC lysis, producing 74% (5C6.10.4) and 68% (6D3.2.10)inhibition. See FIG. 1. FIG. 1 shows inhibition of NCC activity byinhibitor mAbs 5C6.10.4 and 6D3.2.10. MAbs were first purified usingCon-A Sepharose and 10 micrograms was added to each effector: targetcell mixture (160:1 E:T ratio). Values are mean ±SD of three replicates.Asterisks indicate values significantly different from controls(P=0.005). Inhibition by the mAbs was not reversible. Extensive washingof the mAb treated effector cells did not remove any inhibitoryactivity.

Next, non-specific cytotoxic cells were sorted by flow cytometry usingthe FALS and L90.LS parameters. The sorted cells were incubated withmAbs 5C6.10.4 or 2B2.4.9 and tested for lytic activity (FIG. 2). FIG. 2shows inhibition of NCC cytotoxicity by addition of Con-A purified mAbs.Unsorted effector cells are unfractionated anterior kidney cells, whilesorted effector cells were enriched by flow cytometry. The assay wasconducted for 4 hours at 27° C. The number of enriched effector cellsnecessary for higher E:T ratios was not obtainable. Data are mean andS.D. of three replicates at each E:T ratio. Asterisks indicatesignificant difference from sorted controls (P=0.001). 10 micrograms ofmAb was added to each reaction mixture. Significant (96%) inhibition oflysis of NC-37 target cells was produced at a 40:1 E:T ratio in thepresence of 10 micrograms of purified mAb, as well as at a 20:1 E:Tratio.

Effector NCC next were treated with each mAb to determine if inhibitionof conjugate formation occurred. Percoll purified NCC (1.5×10⁶ cells)were treated with 75 or 150 μg of each mAb, the cells were washed andNC-37 target cells were added (1:2 E:T ratio). Compared to NMS (negativecontrol), significant inhibition of conjugate formation was produced byboth inhibitor mAbs, but not by the non-inhibitor mAbs. A dose responsefor mAb inhibition of conjugate formation was also observed.Non-inhibitor mAbs had no significant effect on the percent conjugatesformed. Cytotoxicity experiments conducted indicated that lysis of thetarget cells was likewise inhibited by both concentrations of 5C6.10.4and 6D3.2.0 (Table II).

                  TABLE II                                                        ______________________________________                                        Effects of mAb treatment on NCC conjugate formation with                      NC-37 target cells.                                                                           Percent Inhibition                                            Monoclonal            Conjugate                                               Antibody Treatment.sup.c                                                                            Formation Cytotoxicity                                  ______________________________________                                        Media.sup.a                                                                            --           0         0                                             NMS.sup.b                                                                              (1:200)      0         0                                             5C6.10.4  75 μgm   47.28*    52.87*                                        5C6.10.4 150 μgm   82.40*    84.18*                                        6D3.2.10  75 μgm   46.20*    48.50*                                        6D3.2.10 150 μgm   80.22*    81.15*                                        2B2.4.9   75 μgm   20.87     11.80                                         2B2.4.9  150 μgm   8.80      5.73                                          6D3.4.4   75 μgm   3.27      7.10                                          6D3.4.4  150 μgm   23.10     22.20                                         5C6.10.4.sup.d                                                                         150 μgm   78.05*    81.82*                                        ______________________________________                                         .sup.a Media controls contained 34.6 cells bound to NC37 targets of 300       nontarget cells counted. At an 80:1 ratio, media controls had 38%             cytotoxicity by .sup.51 Cr release assay.                                     .sup.b A 1:200 dilution of NMS produced 30.3 cell/NC37 conjugates of 300      nontarget cells counted. At an 80:1 E:T ratio, NMS controls had 29.7%         cytotoxicity.                                                                 .sup.c All mAbs were purified by immunoaffinity chromatography as             described in the Materials and Methods section. The mAbs were present         during the conjugate assay. Monoclonal antibodies were used at either 75      or 150 μgm/1.5 × 10.sup.6 cells.                                     .sup.d Positive control consisted of ammonium sulfate purified mAb (e.g.,     not subjected to immunoaffinity chromatography).                              *Significantly different from NMS control (p ≦ 0.05) (oneway           ANOVA).                                                                  

FIG. 3 shows inhibition of the cytolytic activity of fresh and activatedNK cells by anti-NCC monoclonals. Endogenous and IL2-activated NK cellswere purified. Each effector cell population was preincubated with theindicated dose of mAb for 1 h at 4° C. (and washed 2×) prior to additionof the K562 target cells. Cytolytic assays were carried out for 3 hr.Percent specific lysis data were converted to LU₂₀ and are indicated inparentheses for each curve. Similar results were obtained in two otherindependent experiments.

All mAbs next were examined for binding to anterior kidney and spleentissue. The fluorescence histogram of mAbs 5C6.10.4 and 6D3.2.10 bindingto anterior kidney cells shows two binding populations, with theconcentration of mAb 5C6.10.4 being approximately 10 times higher thancells stained with mAb 6D3.2.10. The same mAb, 5C6.10.4, binds to cellsfrom two different tissues (spleen and anterior kidney). Cells from bothof these tissues bound approximately the same amount of mAb. Percentspecific staining by each mAb to anterior kidney, spleen and peripheralblood varied according to cell type and mAb. Higher concentrations of6D3.4.4 bound to cells in each tissue compared to any other mAb. Also,spleen cells bound higher concentrations of each mAb compared toanterior kidney and peripheral blood. Peripheral blood did not bindsignificant amounts of any mAb.

In assays using unsorted fish anterior kidney cells as effectors, themAb 5C6.10.4 inhibited NCC lytic activity by 63% (FIG. 2). Similarlevels of inhibition were observed when either the NCC were preincubatedwith the mAbs or when the mAbs were added to the cytotoxicity assaywithout preincubation. Other studies have been conducted using mammalianNK cells to identify inhibitory mAbs with similar properties of theanti-NCC mAbs. NK inhibitory mAbs were effective when addedsimultaneously with the effectors and target. Mammalian NK activity canlikewise be inhibited by pretreatment of the effectors alone with themAb prior to assay.

Other assay conditions were examined to better understand the mAbrequirements to produce inhibition. As little as 10 μg of purifiedanti-NCC mAb produces 96% inhibition of cytotoxicity. In a similar studyin mice a mAb which inhibited Con-A induced T cell proliferation alsoinhibited T cell mediated cytolysis by 67% at mAb concentrations as lowas 1 mg/ml.

The mechanism(s) of mAb inhibition of NCC was determined b investigatingthe stage of the lytic cycle at which inhibition occurred. Pretreatmentof the NCC with purified mAbs produced significant decreased conjugateformation between NCC and NC-37 targets. Although binding occurs betweenthe non-inhibitor mAbs and the effector cells, conjugate formation wasnot significantly reduced. Other work has shown that mAbs specific forCD45 (T200 family) and CMRF-11, CMRF-12 and CMRF-26 all inhibited NKactivity at a post binding stage. Unlike these results, the mAbs of thepresent invention were shown to react against an antigen bindingreceptor on NCC. This clearly demonstrated their ability to inhibit thefirst stage (conjugate formation) of several sequential steps that leadto target cell lysis.

To evaluate the relative concentrations of the membrane determinantsfound on NCC and to determine the distribution of receptor bearing cellsin fish lymphoreticular tissue, FCM analysis was conducted. Bothinhibitor and non-inhibitor mAbs bound to anterior kidney and spleencells. NCC were not detectable in peripheral blood by FCM analysis,however. It has been found that there is 16-18% conjugate forming NCC inanterior kidney cells in the presence of NC-37 target cells. By FCManalysis, from 25% (5C6.10.4) to 39% (6D3.4.4) of anterior kidney cellscontain the determinants recognized by these mAbs. These differencesobserved between the single cell in agarose technique and the FCManalysis data obviously reflect the sensitivities of these protocols fordetecting NCC.

Isolation and Characterization of the NK Cell Receptor. NCC were sortedby FCM and detergent solubilized extracts of these cells were purifiedby affinity chromatography (Affigel 15) using mAb. The same dimer waspurified using either inhibitory (6D3.2.10) or non-inhibitory (6D3.4.4)mAbs. Both the inhibitory and noninhibitory mAbs precipitated 2proteins, one with a molecular weight of approximately 41,000 D and asecond of approximately 38,000 D. This complex consists of 41,000 and38,000 D molecules which co-migrate as two separate chains regardless ofthe presence of nonreducing or reducing agents. Molecules with thischaracteristic have not been reported previously for any type ofeffector of "non-specific" cytotoxicity (e.g., NK, anomalous killer,promiscuous killer, LAK, CTL-NK, etc.).

Similar results were obtained with immunoprecipitation experiments usingmAb 5C6.10.4 and analyzed by SDS-PAGE under reducing conditions. Theprecipitated 43,000 D and 38,000 D molecules were similar to thoseobserved with affinity chromatography.

The proteins and antibodies of the present invention have uses inaddition to providing information regarding their origin and function.They can be used in assays to screen for related or altered proteins.They can be used, alone or in combination with biologically activecompounds, for treatment of disease states including cancer andautoimmune diseases. Such uses are intended to come within the scope ofthe present invention. The proteins of the present invention, both theantigen and the receptor, can be further characterized by studiesinvolving the extent and function of carbohydrate on the molecules, therate of turnover on the cell surface, the isoelectric points, therelationship and homology between the dimer subunits, and the effect ofmodifications thereof on biological function. Modifications to theproteins chemically as well as by production of mutants can be used toachieve the latter. Methods to produce this information are available tothose skilled in the art and are described in detail herein.

Since the proteins have been purified to homogeneity as measured bySDS-PAGE, it is possible to sequence the molecules so as to compare thesequences with known molecules and to prepare nucleotide probes forscreening of libraries for sequences encoding the molecule. The probesare also useful in measuring mRNA content in the various low and highexpression cell lines and mutants to determine at what level, mRNAprocessing or protein assembly, that the changes occur. Alternatively,the clones can be screened using the mAbs described above orheterologous antisera raised to the purified proteins. cDNA librariesare available from a number of cell lines. They can also be prepared asrequired using an expression vector such as lambda gt11 using poly(A)RNA isolated from effector or target cell lines. Positive clones can berescreened to homogeneity at low density and compared with one anotherby restriction mapping and Southern blot analysis to identify identicalor overlapping clones. The sequencing data from the identified clonescan be used to provide further support and means for identification ofthe genes for either the receptor or the antigen, and aid inidentification of related molecules. Further, these sequences and probescan be used in assays to characterize immune cells and target cellsaccording to their receptor or antigen. As importantly, identificationand characterization of the sequences and their respective functionprovides a means for modifying relationships in vivo, either enhancingor inhibiting the cytolytic activity of natural killer cells againsttarget cells or even cells which would not normally serve as targets forthe natural killer cells. The sequences could also be used to map thelocation of the genes for these proteins on the chromosomes, allowingscreening for potential defects in the immune response, eithercongenital or disease or environment induced. Knowledge of the genesencoding these molecules also provides information on the regulation ofexpression and function of these proteins, information not presentlyavailable for any molecule within the immune system which is common toso many different species of such wide evolutionary divergence. Methodsto accomplish these ends are presently available to those skilled in theart once the proteins and sequences or probes for sequences encodingthese proteins are known. The sequences, probes, and uses therefore areintended to come within the scope of the present invention.

MATERIALS AND METHODS:

Animals. Outbred channel catfish (Ictalurus punctatus) of both sexes,weighing 10 to 25 grams, approximately 6 months to one and one-halfyears of age were obtained from local commercial farms. Fish weremaintained in 570 liter fiberglass running streams at temperaturesranging from 16°-19° C. The diet consisted of pelleted fish feed (PurinaCatfish Startena, Ralston-Purina Co.). Balb/c mice were used for mAbproduction and were purchased from Harlan Sprague-Dawley (Indianapolis,Ind.).

Media. Cell cultures were maintained in RPMI-1640 (Flow Laboratories,Rockville, Md.) adjusted to an osmolarity of 250 milliOsmoles/kg H₂ Ousing a micro-Osmette (Precision Systems, La Jolla, Calif.).

Preparations of Cell Suspensions. Fish were lightly anesthetized withethyl-aminobenzoate (Sigma Chemical Co., St. Louis, Mo.) in water andsacrificed. Anterior kidney, peripheral blood and spleen cells wereremoved and single cell suspensions prepared, as described by S. S.Graves et al., Dev. Comp. Immunol. 8, 293 (1984), the teachings of whichare incorporated herein. Cells were washed twice (200 ×g; 10 min),stained with Trypan blue and counted in a hemocytometer. Samples forflow cytometric analysis were adjusted to 2×10⁶ total cells/ml.Erythrocytes were removed by preparing a solution of phosphate-bufferedsaline (250 milliOsmoles/kg H₂ O) and Percoll® (Pharmacia FineChemicals, Inc., Uppsala, Sweden). This solution was added in 2 mlvolume to 15 ml plastic tubes. A suspension of anterior kidney,peripheral blood or spleen cells (3×10⁸ cells/ml) was layered in 1 mlvolumes on the Percoll® solution. The tubes were centrifuged (300×g for10 min) and the leukocytes withdrawn from the interface and washed (2×).For effector cell preincubations, cells were suspended at 2×10⁷ cells/mlin RPMI-1640 (10% FBS) and incubated for 4 hr in 24 well tissue cultureplates (Linbro Plastics, Flow Laboratories, McLean, Va.) at 26° C. and5% CO₂ tension.

Production of Monoclonal Antibody Directed Against Receptor Protein. Themyeloma cell line P₃ X63-Ag⁸ (P₃) was cultured in complete RPMI-1640media containing 10% heat inactivated FBS. Spleens were asepticallyremoved from mice previously immunized with FCM sorted NCC, as describedby D. L. Evans et al. in Dev. Comp. Immunol. 11, 95 (1987), theteachings of which are incorporated herein on target cells, as describedbelow, and washed (2×) in RPMI-1640 (without serum). Spleen cellsuspensions were mixed with P₃ cells (logarithmic growth). Cells werewashed (3×) counted and mixed at a ratio of 5.0×10⁶ spleen cells to1.0×10⁶ myeloma cells. Cell fusion was accomplished using 1.0 ml ofpolyethylene glycol (BMB Biochemicals Indianapolis, Ind.). After thefusion step, cells were centrifuged (220×g, 10 min), the supernatant wasremoved and the cells resuspended in 40 ml of complete RPMI (15% FBS).The cell suspension was divided equally (2.5×10⁶ cells/ml) into thewells of a 48-well plate and incubated overnight at 37° C. in 5% CO₂tension. The media was then changed to complete RPMI-1640 supplementedwith 15% FBS and 0.1 mM hypoxanthine, 4.0×10⁻⁷ M aminopterin and1.6×10⁻⁵ M thymidine (HAT medium). The HAT medium was replenished (50%substitution by volume) on day 7 after the fusion. On day 10 the HATmedium was replaced by complete RPMI medium supplemented with 0.1 mMhypoxanthine and 1.6×10⁻⁵ M thymidine (HT medium). Culture medium wasthereafter replenished as necessary. Supernatants containing growinghybridomas were then screened by ELISA.

NCC Solubilization for Receptor Isolation. Non-specific cytotoxic cellswere washed twice in PBS (pH 7.5), resuspended at 5×10⁸ cells/ml, andsolubilized by addition of 5.0 ml of 20 mM Tris-HCl at pH 7.5,containing 1 mM phenylmethylsulfonyl fluoride and 1% Nonidet-P40. Aftertwo hours (4° C.) the sample was centrifuged (100,000×g; 60 min) and thesupernatant dialyzed against PBS for 48 hrs (4° C.) and stored at -20°C. Cell extracts (7 micrograms) of each sample were electrophoresed in12.0% SDS-PAGE; 5 hr at 15 milliamps. Gels were stained using a standardsilver nitrate technique (Bio Rad Laboratories, Richmond, Calif.).

Isolation of Anti-Receptor Antibodies. Hybridomas from the fusionsbetween P₃ myeloma cells and immune spleen cells from Balb/c miceimmunized with FCM purified NCC were screened using an ELISA technique.The ELISA plates were coated with NCC preparations obtained fromanterior kidney tissue. For this, NCC were used which had been enrichedapproximately 5-6× on 45.5% Percoll®. ELISA positive hybridomas weretested for inhibition of NCC lysis of ⁵¹ Cr-labeled NC-37 target cells.Of the ELISA positive mAbs tested, four mAbs were chosen for furtherstudy. These were designated 5C6.10.4, 6D3.2.10, 6D3.4.4, and 2B2.4.9,respectively. All four mAbs are IgM and all were subcloned two times bylimiting dilution.

Concanavalin A Sepharose Chromatography of mAbs. IgM monoclonalantibodies were purified by affinity chromatography according to themethod of M. D. P. Boyle et al. in J. Immun. Methods 32, 51 (1980)utilizing Con-A Sepharose beads (Affigel Con-A, BioRad Labs, Richmond,Calif.). The Concanavalin A Sepharose gel was washed with 3-5 bedvolumes of sample application buffer (containing 10 mM Tris [pH 7.2], 1mM Mg⁺⁺, and 1 mM Ca⁺⁺). After removing excess buffer above the gel, 2-5ml of sample was applied and mixed very gently, forming a slurry. Thecolumn was then washed with the application buffer, and samplesmonitored until the 0.D. (280 nm) of the effluent was the same as theapplication buffer. The specifically bound IgM mAbs were then elutedwith 200 mM alpha-D-methyl mannopyranoside. The gel was regenerated bywashing with 3-5 bed volumes of the application buffer, and was storedat 4° C.

Cytotoxicity Assay. Cytotoxic activity was measured using a ⁵¹Cr-release assay as previously described by Graves et al., (1984). TheNC-37 human lymphoblastoid B cell line was used as a target. These cellswere maintained in RPMI-1640 (10% FBS) at 37° C. and 5% CO² tension.Target cells were labeled with 100 μCi of ⁵¹ NaCrO₄ (AmershamCorporation, Chicago, Ill.) for 2 h at 37° C. as described by Graves etal. Fish NCC were assayed using head kidney cells prepared as singlecell suspensions, washed twice, counted, and incubated for 4 h at aconcentration of 2×10⁷ cells/ml in RPMI 1640 containing 10% FBS.Following this preincubation period, NCC were harvested, centrifuged,and added to ⁵¹ Cr-labeled target cells at different effector:targetcell ratios. To assess cytotoxicity, 100 microliters of supernatantswere harvested from each well and radioactivity was determined in aBeckman Biogamma II gamma counter. Cytotoxicity was expressed as percentspecific release (% SR) and calculated using the following formula:##EQU1##

Flow Cytometric Analysis. An EPICS V® 753 flow cytometer (CoulterElectronics, EPICS Division, Hialeah, Fla.) was used to analyze cellsize, granularity, and fluorescence. Samples of viable cells wereprepared at a concentration of 2×10⁶ cells/ml and analyzed by forwardangle light scatter (FALS), Log₁₀ 90° light scatter (L90° LS), or greenfluorescence. The instrument was standardized daily using Fulbright (GRII) 9.75 micrometer diameter fluorescent polystyrene microspheres andoperated at constant laser power and photomultiplier settings. The greenfluorescence PMT received light after passage through a 488 nm laserblocker and a 525 nm bandpass interference filter. Analyses wereperformed using a flow rate of 300-400 cells/second. The FALS and L90°LS parameters were used to electronically gate the lymphocyte populationto exclude debris and to enable analysis of negative cells. All analysiswas done using an argon-ion laser (488 nm emission) at a constant powerof 500 mW.

For all sorting, the sheath buffer (normal saline) was adjusted to 250mOsm/kg H₂ O and cells were sorted based on previously establishedparameters of FALS and L90° LS. Fluorescence analysis was accomplishedusing FITC conjugated anti-mouse IgM antibodies (Sigma Chemicals, St.Louis, Mo.). Cells were excited at 488 nm and fluorescence was detectedusing a 590 nm (short pass) dichroic mirror and a 525 nm band passinterference filter. Two different electronic gating procedures wereused: for analysis of percent specific binding, FALS and L90° LS gatingwas used; and to obtain optimal histograms, gating was done based on theLOG₁₀ green fluorescence signal.

To determine mAb binding, cells were harvested from the anterior kidneyand spleen as previously described and resuspended at 1 to 2×10⁶ /ml inRPMI media. Each monoclonal antibody to be tested was added (100micrograms) to 0.5 ml of the cell suspension and incubated 30 min (4°C.). Normal mouse serum or irrelevant IgM monoclonals were used ascontrols. Cells were washed two to three times in cold (4° C.)RPMI-1640. Fluorescein isothiocyanate conjugated anti-IgM or anti-mouseIgG (Sigma Chemical Co., St. Louis, Mo.) (1:20 dilution) was added in100 μl. The cells were again incubated on ice for 30 min, washed twicein cold (4° C.) media and resuspended in 1 ml media for flow cytometricanalysis.

Conjugate Assay. To prepare cells for the assay, anterior kidney cellswere isolated, washed, and fractionated by layering over 45.5% Percoll®(3 ml of cells on 6 ml Percoll). After centrifugation for 20 minutes at200 x g, the cells at the media--Percoll interface were harvested,washed and counted. 70×10⁶ cells/ml were then incubated for 6 hours (25°C.).

To prepare Percoll purified NCC for the conjugate assay, 7.5×10⁵ cellswere dispensed into each tube and appropriate media, NMS or purified mAbwas added (total volume of 375 μl). The cells were incubated for 60minutes (5% CO₂, 25° C.), centrifuged (200×g) and resuspended in 375 μlof RPMI-1640.

The single-cell in agarose conjugate procedure was conducted aspreviously described for fish NCC by Evans et al. (1984). Briefly, 100μl of treated NCC (2×10⁶ cells/ml) and 200 μl of NC-37 (2×10⁶ cells/ml)cells were mixed in round bottom glass tubes and incubated for 5 minutes(30° C). The cells were then centrifuged for 5 minutes (200×g), thepellet gently resuspended into 50 μl of premelted agarose and then themixture poured onto agarose precoated slides. To determine percentconjugates, 300 effector cells were counted and the number of effectorsbound to NC-37 cells was determined. Coded samples were counted(single-blind analysis).

Affinity Chromatography of Receptor. For purification of the receptor,Con-A Sepharose purified monoclonal antibodies 6D3.2 and 6D3.4.4 werecoupled with Affigel 10 ™ (Bio Rad, Richmond, Calif.) at a concentrationof5 mg of protein/ml of gel for 4 hr at 4° C. with gentle shaking in 10mM Hepes buffer (pH 7.5). The reaction was stopped by centrifugation andthe gel was resuspended in 0.1M glycine ethyl ester (pH 8) for one hr toblock the remaining active sites. After repeated washes with PBS (pH7.5), the gel was stored at 4° C. Solubilized NCC lysates were incubatedwith 6D3.2 and 6D3.4.4 affinity gels at a concentration of 2 mgprotein/ml of beads overnight at 4° C. with gentle shaking. The gelswere poured into two columns and washed extensively with PBS (pH 7.50)until eluted proteins could not be detected at OD₂₈₀. Specific effectorcell (NCC) antigens were eluted with glycine/HCl buffer (0.1M adjustedto pH 2.5 with 0.2M HCl) until all protein was eluted. Fractions werecollected into tubes containing 100 μl of 1M Tris HCl, pH 8.0. The peakfractions were pooled, dialyzed against PBS, concentrated usingcarboxymethyl cellulose and stored at -20° C.

Immune Precipitation and SDS-PAGE Analysis of the NCC Receptor. Fish NCClysates were precleared with a 12 hour incubation with Sepharose CL4Bbeads at 4° C. The precleared lysate then was incubated with Protein ASepharose 4B bound to mAb 5C6.10.4 for 4 h at 4° C. The ProteinA-Sepharose 4B complex was washed (3×) with 1% Triton X-100 buffer. Thespecifically bound molecules then were eluted with 2× sample buffer (125mM Tris-HCl, pH 6.8 with 20% glycerol (v/v), 4% SDS (w/v)) and 10%beta-mercaptoethanol (v/v) and samples were electrophoresed (11%SDS-PAGE). The gels were stained by the silver stain method (Bio RadLaboratories, Richmond, Calif.).

Statistical Analysis. Probability statements were obtained by one-wayanalysis of variance (ANOVA).

Immunoprecipitation of the Receptor. Labeled cells are resuspended inbuffer to 2-4×10⁷ /ml. An equal volume of lysis buffer (1% NP40; 1% BSA;1 mM PMSF) is added. Nuclei, debris and unlysed cells are removed bycentrifugation at 3,000×g for 15 min. Immune precipitation is carriedout as follows: Labeled cell extract is mixed with the appropriate mAbin a buffer containing 0.01M Tris pH 7.5, bovine serum albumin (100μg/ml) and 0.3% Triton X-100. Incubation is for 3 h at 4° C.Staphylococcal protein-A is added (10%, v/v) to the antigen-antibodymixture, and incubated for 1 h at 37° C. This mixture is centrifuged(200×g, 2 min, 4° C.) and the pellet treated by adding 50 μl of SDSbuffer (3% SDS/2% mercaptoethanol/0.05 Tris, pH 6.8/5% glycerol)followed by boiling 10 min and centrifugation. Alternatively, thestaphylococcal protein-A antigen-antibody mixture is suspended in 4% SDSin 8M urea (containing 0.05M Tris, (pH 8.4), boiled 3 min andcentrifuging (2000×g, 6 min). In each immune precipitation, labeledantigen will always be in excess.

Immunoprecipitation and SDS-PAGE Analysis of the NC-37 Target Antigen.NC-37 cells were incubated for 6 hours with 100 μCi/10⁶ cells/ml ³⁵S-methionine (New England Nuclear, Boston, Mass.). The labeled cellswere centrifuged and lysed with 1% Triton X-100 buffer (0.15M NaCl, 1%NaDOC, 1% Triton X-100, 10 mM Tris, pH 7.4, 1 mg/ml BSA, 0.02% NaN₃, 0.1mM PMSF). Nuclei were removed by centrifugation and the supernatant wasprecleared (12 h) with Sepharose CL 4B beads (4° C.). The preclearedlysate then was incubated with Protein A Sepharose 4B bound to mAb18C2.8.3 or 1E7. The Protein A Sepharose 4B complex next was centrifugedand washed (3×) with the above buffer, the mAb purified target cellantigens were eluted with 2× sample buffer (125 mM Tris-HCl pH 6.8 with20% glycerol v/v, 4% SDS w/v and 10% beta-mercaptoethanol v/v) andsamples were electrophoresed (11% SDS-PAGE). The gels were dried andexposed to X-ray film (X-OMAT, KODAK, Rochester, N.Y.) for 5 days atroom temperature.

Immuneprecipitation of the NK Cell Antigen was performed as for thereceptor.

Phycoerythrin-B Conjugation. Thiolated phycoerythrin is prepared by theaddition of 600 μl of 15.5 mg/ml iminothiolene hydrochloride (SigmaChemical Co.) to 1.2 ml of 3.6 mg/ml phycoerythrin-B in 125 mM sodiumphosphate (pH 6.8). After 90 min at room temperature, the reactionmixture is dialyzed overnight at 4° C. against 50 mM sodium phosphate(pH 6.8) and then for 2 days against pH 7.5 buffer (this gives anaverage of 8-SH-groups/molecule).

A 30 μl aliquot of 1.1 mg/ml N-succinimidyl3-(2-pyridylthio-proportionate (SPDP) (Pharmacia Fine Chemicals,Piscataway, N.J.) in ethanol is added to 700 μl of 4.2 mg/mlimmunoglobulin in 50 mM sodium phosphate (pH 7.5). The molar ratio ofSPDP to Ig is 5.3. The reaction is allowed to proceed for 2.5 h at roomtemperature. Thiolated phycoerythrin (400 μl of 1.7 mg/ml in the samebuffer) is added to 500 μl of the reaction mixture. The molar ratio ofactivated Ig to thiolated phycoerythrin-B is 4.7. After 12 h at roomtemperature, 100 μl of 80 mM sodium iodoacetate is added to block anyremaining sulfhydryl groups.

Phorbol Ester Treatment. Phorbol-12-myristate-13acetate (PMA) and itsanalog 4-alpha-phorbol-12, 13-didecanoate (4-alpha PDD) (an inactiveanalog of PMA), is used to determine the effects of phorbol esters onNCC/NK lytic activity. NCC and NK cells are treated with PMA (10⁻⁶ M) orwith 4 alpha PDD (10₋₆ M). Cells are treated from 1 h to 40 h todetermine the effects of these substances on effector cell activity, andon the membrane expression of the effector cell receptor. Increasedexpression of the monoclonal reactive determinants is determined by FCMand by increased (or decreased) lysis of susceptible target cells.

Radiolabeling of Surface Carbohydrate. Sialic acid residues ofglycoproteins are labeled by tritiated sodium borohydride, NaB³ H₄,reduction of oxidized carbohydrates. One to two×10⁷ lymphocytes in 1 mlof PBS are incubated for 10 min at 0° C. with 1 mM sodium periodate forthe formation of aldehyde groups on the sialic acid residues. Thereaction is stopped by the addition of glycerol to a final concentrationof 25 mM and the cells are washed twice with PBS. The pelleted cells areresuspended in 0.5 ml of Dulbecco's balanced salt solution (DBSS) and 1mCi of NaB³ H₄ is added to reduce the aldehyde groups to theircorresponding alcohols. The mixture is incubated for 30 min at roomtemperature and washed twice with DBSS.

Human NK Cells. Heparinized peripheral blood is obtained from normaldonors (aged 18-30 years) as a source of NK cells. Peripheral bloodlymphocytes (PBL) are centrifuged over Ficoll-Hypaque gradients astaught by Boyum, Scan. J. Clin. Lab. Invest. 21(Suppl. 97), 9 (1968) toobtain mononuclear cells (MNC). MNC are enriched for NK activity by oneor a combination of the following techniques: (a) MNC are plated onplastic petri dishes for 2 cycles of 1 h at 37° C. and the nonadherentcells carefully collected (PNAd; Fischer et al., Cell. Immunol. 58, 426(1981); (b) PNAd are passed over nylon wool columns and nonadherentcells collected according to Julius et al., Eur. J. Immunol. 3, 645(1973); (c) PNAd are centrifuged over Percoll gradients according to themethod of Storkus and Dawson, J. Leukocyte Biol. 39, 547 (1986); and (d)PNAd are panned over mAb PKT3 coated petri dishes and the nonadherentcells collected. Cell viability is assessed by Trypan blue dyeexclusion. The various NK populations are phenotyped for the NK markersLeu 11, Leu 7, Leu 19 by flow cytometry.

Culture and Cloning of NK Cells. NK cells isolated by the above methodsare the source of cells for bulk culture and cloning. Bulk cultures aregrown in 25 mm² flasks and culture growth initiated by stimulation witheither lectins, IL2, irradiated tumor cells or a combination of thesestimuli. Irradiated feeder cells are also added. Cultures are maintainedby the weekly addition of IL2 alone. The original stimuli and feedercells are re-added only if growth of the cultures declines. Irradiated Bcell lines have been found to serve as feeder cells for human NKcultures. Cultures are screened weekly for phenotype and only typical NKcultures maintained. NK cells are cloned by limiting dilution at 0.25cells/well in 96 well microtitre plates as described by Van de Griend etal., J. Immunol. Methods 66, 285 (1984). The clones are expanded asnecessary to 25 mm² flasks and periodically screened for theirphenotypes. Up to 100×10⁶ cells can be obtained per clone with thisprotocol.

Stimulation of NK for Lymphokine Production. 1-2×10⁶ NK cells/well areplated in 1.0 ml of complete medium in 24 well Costar plates. Theappropriate stimuli are added and the reactions incubated for 24 h at37.C. Afterwards the wells are harvested and the cells removed bycentrifugation (1100×g, 10 min). The supernates are sterilized byfiltration (0.2μ, Millipore) and stored at 4° C. until analysis. Thefollowing stimuli are utilized: IL-2 (30 units/ml), Con-A (5 μg/ml), PHA(1 μg/ml), and antigen tumor cells, 1:1 NK to tumor cell ratio, attachedvia poly-1-lysine to the wells and fixed with 0.025% glutaraldehyde for5 min at 23° C. Complete medium alone serves as a control forspontaneous lymphokine secretion.

Flow Cytometric Analysis of Cellular DNA Content and Distribution.Analysis of DNA content and distribution following of NK cellsexperimental will be done. Washed and resuspended NK cells (1×10⁶cells/ml in PBS containing 10 μg/ml of the DNA-binding dye Hoechst 33342and 0.1% Nonidet P-40 detergent will be analyzed using afluorescence-activated cell sorter. For this, the laser is adjusted toemit 50 mW at 351 and 363 nm. Fluorescence is detected without anyintervening optical filters. Narrow angle forward light scatter is usedto identify viable cells. G₀ /1, S, and G₂ +M populations are identifiedon the basis of fluorescence intensity. A semiquantitative method isused to estimate the relative number of cells in the different phases ofthe cell cycle (Harris and Sekaly, J. Immunol. 133(1), 40 (1984). The G₁and G₂ +M phase populations are assumed to be normally distributed andthe mean fluorescence intensities determined directly from the DNAdistribution. Standard deviations (SD) are calculated by dividing thefull width at half-maximum of each peak by 2.35. The G₁ population isdefined as the region of DNA distribution within 2 SD above and belowthe mean fluorescence intensity of the G₁ peak. The G₂ +M population issimilarly ascribed to the region of distribution within 2 SD of the G₂+M peak. S-phase cells are defined as having fluorescence intensitiesdistributed between the G₁ and G₂ +M regions.

NK Radioisotope Labeling and Experimental Protocols. Changes inphospholipid metabolism after stimulation of the NK are determined asfollows. NK are collected, prepared and then washed once with labelingbuffer (137 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl₂, 1.0 mM CaCl₂, 20 mMHEPES, 25 mM glucose, 1 mg/ml bovine serum albumin, pH 7.4), resuspendedat 20×10⁶ cells/ml in

labeling buffer and prelabeled with 50 μCi/ml ³² p-orthophosphate(Amersham, 200 mCi/mMol) for 1-2 h at 37° C. in order to equilibrate thecellular ATP pools. At the same time the cells are prelabeled with 20μCi/ml ³ H-myo-inositol (Amersham, 15 Ci/mMol) as a tracer for theidentification of the various inositol lipid species. At the end of theprelabeling time period the cells are either washed twice with labelingbuffer and resuspended to 6×10⁶ cell/ml in the same buffer (forshort-term assays), or are immediately diluted to 6×10⁶ cells/ml withlabeling buffer containing additional radioisotopes (³² P and ³H-inositol) to prevent depletion of ³² P-labeled ATP and trace-labeledinositol pools (for long-term assays). All reactions are carried out in0.5 ml aliquots (1.5×10⁶ NK total) at 37° C. Short-term reactions areperformed in polypropylene tubes using a 37° C. water bath. Long-termreactions are performed in polypropylene tubes, except for antigenstimulus in which the tumor cells were attached to poly-1-lysine-treatedwells of a 12-well Costar plate and glutaraldehyde-fixed prior to theaddition of the NK) in a humidified 37° C. CO.sub. 2 incubator. Allstimuli are added to the NK while on ice. Reactions are initiated byplacing the NK at 37° C., except for short-term antigenic stimulation inwhich the NK and tumor cells are centrifuged together for 2 min at 1000rpm, 4° C. to initiate conjugate formation immediately prior to beingplaced at 37° C. Reactions are terminated by the addition of 1.95 ml ofchloroform/methanol/conc. HCl (100:200:1, v/v) with immediate vortexingof the samples.

To analyze for kDAG generation the cells are labeled for 24 h with 1μCi/ml ³ H-arachidonic acid (Amersham, 130-200 Ci/mmole). The NK areprepared and the reactions are initiated and terminated as describedabove for phospholipids analysis.

For IP analysis the NK (20×10⁶ /ml) are incubated for 4-5 h at 37° C. inlabeling buffer containing ³ H-myo-inositol (14-20 Ci/mmol) at aconcentration of 10 μCi/ml. Afterwards NK are washed twice in labelingbuffer and resuspended to a final concentration of 60-80×10& NK per mlin the same buffer (4° C.) containing lithium chloride (10 mM).Reactions are initiated as described above and terminated by theaddition of 1 ml of a 11.25% solution of trichloroacetic acid (TCA) at4° C. The samples are mixed by vortex and held at 4° C. for 20 min priorto storage at -70° C.

Analysis of NK Lipid Metabolism. NK lipids are extracted by the furtheraddition of 0.65 ml chloroform and 0.65 ml 0.1N HCl to the chloroform/methanol-terminated reactions mixtures. The reaction samples are mixedby vortex and centrifuged (2000 rpm, 10 min, 4° C.) prior to collectionof the organic layers. The samples are then re-extracted with 1.0 mlchloroform and the two organic phases are combined and stored at -70° C.until analysis. Prior to thin layer chromatographic (TLC) analysis thesamples are evaporated to dryness at 37° C. under nitrogen. TLC analysisis performed using 20 cm×20 cm, 0.2μ silica plates (Silica gel 60,Merck)which have been preactivated for 1-2 h at 110.C immediately prior touse. The dried samples are dissolved in 10 μl of chloroform and appliedto the TLC plates. This procedure is then repeated for each sample. 90%or more of the total radioactivity per sample is transferred to the TLCplate. The plates are then reheated at 110° C. for 10 min prior todevelopment.

The lipids are analyzed by ascending TLC in each of the followingone-dimensional systems. System I consists ofchloroform/methanol/petroleum/ether/ glacial acetic acid/boric acid(48:24:36:12:2.16, v/v/v/v/w) which separates the following lipidclasses in ascending order: lysophosphatidylcholine (LPC), sphingomyelin(SM), phosphatidylcholine (PC), phosphatidylinositol (PI),phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidicacid (PA), free fatty acids (FFA) and triglycerides (TG). System IIconsists of chloroform/acetone/methanol/glacial acetic acid/water(40:15:13:12:8, by volume) and utilizes 1% di-potassium oxalate (inmethanol/water, 2:3)-treated TLC plates. System II resolves thefollowing lipid classes in ascending order:phosphatidylinositol-4,5-biphosphate (PIP₂),phosphatidylinositol-4-monophosphate (PIP), PI+SM, PS+PC, PE, and PA.System III consists of hexane/diethylether/formic acid (90:60:12, v/v/v)and separates (also in ascending order): phospholipids (origin),monoglycerides (MG), 1,2-DAG, 1,3-DAG, FFA, TG and cholesterol esters.For systems I and II, which are used to separate the ³² P-labeledphospholipids (³ H-inositol radioactivity is used to assess thecross-contamination of lipid classes), between 5000 and 50,000cpm/sample (depending on experiment) are spotted for analysis. Forsystem III, which is used for DAG analysis, between 5×10⁵ and 1×10⁶ cpmof ³ H-AA labeled lipids will be spotted per sample. The lipids arevisualized by exposure to iodine vapors and each individual lipidscraped into a scintillation vial. Lipids are extracted from the silicaby the addition of 10 ml of Aquassure (New England Nuclear) per tubewith vigorous vortexing. Radioactivity is then determined byscintillation counting with correction for quenching.

Phospholipid analysis is performed by comparing cpm of treated CTL tocpm of control, unstimulated CTL. Controls are performed for eachtimepoint, allowing the data to be presented as change vs. control. DAGis calculated as a percent of total lipids and data is presented as theratio of stimulated CTL to control, unstimulated CTL for each conditionand each timepoint.

Analysis of NK Inositol Phosphates. The TCA-diluted samples (stored at-70° C. until analysis) are allowed to thaw without exceeding 4° C. andthen mixed by vortex prior to centrifugation for 10 min at 3000 rpm, 4°C. Aqueous supernates, containing ³ H-myo-inositol and itsphosphorylated derivatives (glycerolphosphorylinositol, IP, IP₂ andIP₃), are separated from the pellets and extracted five times with 2.4ml of diethylether. Following neutralization to pH 7.0 with 6.25 mMsodium tetraborate, the supernates are applied to anion exchange columnsconsisting of 1.2 ml of Dowex 1-8 100-200 mesh (formate form, SigmaChemical Co.) and sequentially eluted as described by Imboden and Stobo,J. Exp. Med. 161, 446 (1985). Inositol and glycerolphosphorylinositolelute together, followed by the sequential elution of IP, P₂ and IP₃.

Analysis of Protein Phosphorylation. The effects of NK stimulation on denovo protein phosphorylation are analyzed by the following procedure. NKare prepared as described, washed twice in buffer B containing 1 mg/mlBSA and resuspended at 20×10⁶ cells/ml in the same buffer. ³²P-orthophosphate is added to 50-100 μCi/ml and the NK prelabeled for30-60 min at 37° C. to equilibrate the cellular ATP pools. At the end ofthis time the NK is processed. For short-term assays (0-30 min), the NKis washed twice and resuspended at 2×10⁶ cells/ml in the buffer. Forlong-term assays (30 min-24 h), the NK is diluted to 2×10⁶ cells/ml withbuffer B without washing and additional radioisotope is added to preventdepletion of the cellular ATP pools. All reactions are initiated byplacing the NK at 37° C. Reactions are terminated by the addition of 3ml of ice-cold PBS containing 10 mM EDTA, 0.1M NaF, 0.1 mM PMSF. Thecells are washed twice in such a fashion and the cellular pellets areresuspended in 100 μl of buffer B containing 0.1M NaF, 0.1 mM PMSF,5×10⁻⁵ M 2-mercaptoethanol, 1% Triton X-100. This reaction is incubatedfor 30 min on ice followed by centrifugation at 2400×g for 10 min at 4°C. to remove the insoluble nuclear pellet. The supernatant is used as asource of detergent-soluble plasma membrane/cytoplasmic proteins.Proteins associated with the nuclear pellet are extracted by a 30 mintreatment on ice with 400 mM NaCl, 5×10⁻⁵ M 2-mercaptoethanol, 2 mMMgCl₂, 0.1 mM PMSF, 0.1M NaF, 20 mM HEPES, pH 7.5, the pellet againcentrifuged and the supernatant collected for analysis.

Two-Dimensional Peptide Mapping. As described by Accolla, J. Exp. Med.157, 1053 (1983), the radiolabeled isolated proteins (from the targetcell or effector cell immunoprecipitates) are cut out of the driedSDS-PAGE gel and eluted in 0.1% SDS in PBS. The eluted material isreduced with 20 mM dithiothreitol for 60 min, and alkylated with 60 mMiodoacetamide for 30 min. Peptic digestion is performed in 100 μl offormic acid/acetic acid/water 1:4:45 (vol/vol) with pepsin in thepresence of bovine serum albumin carrier (1:50 enzyme/protein ratio) for16 h at 37° C. Two-dimensional peptide mappings is performed on silicagel plates. Electrophoresis is performed in the first dimension, withchromatography performed (at right angles) in the second dimension inn-butanol/acetic acid/water/pyridine) 75:15:40:50 (vol/vol).

Selection of Cell Loss Variants. Mutagenesis of the cells (target oreffector cells) is performed as described by Kavathas et al., Proc.Natl. Acad. Sci. USA 77, 4251 (1980). 5×10⁶ cells is irradiated with20-300 rads, washed and cultured in complete medium at 1×10⁶ cells/ml.After 1 week, the cells are immunoselected by adding a saturating doseof either the anti-target cell or anti-effector cell mAbs andcomplement. The addition of mAb and complement is repeated every weekfor a period of 1 month. Cells surviving the immunoselection (cell lossvariants) are cloned under limiting dilution conditions, as required.

Modifications and variations of the present invention, a dimeric proteincommon to the surface of natural killer cells of mammalian and fishorigin which functions in recognition of target cells, the antigen onthe surface of the cells recognized by the natural kill receptor, mAbsto the receptor and target cell protein, and methods for altering theinteraction between natural killer cells and target cells, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention. Such modifications and variations areintended to come within the scope of the appended claims.

We claim:
 1. A protein isolated from the surface of natural killer cellsof mammalian origin and non-specific cytotoxic cells of fish origin,wherein said protein is a dimer by SDS gel electrophoresis underreducing or non-reducing conditions of a protein molecule having amolecular weight of between about 38,000 and a protein molecule having amolecular weight of about 43,000, said protein dimer non-specificallyinteracting with an antigen or a target cell to activate the naturalkiller cell and to induce lysis of the target cell by the activatednatural killer cell.
 2. The protein of claim 1 wherein lysis of targetcells bearing an antigen recognized by the natural killer cells can beinhibited by binding of a second molecule to the protein present on thesurface of both natural killer cells of mammalian and non-specificcytotoxic cells of fish origin.
 3. The protein of claim 1 wherein saidprotein is functionally and immunologically distinct from thetransferring receptor, the antigen specific cytolytic thymus derivedlymphocyte receptor, the IgG Fc receptor, and the laminin receptor. 4.The protein of claim 3 wherein the inhibitory molecule is an antibodyimmunoreactive with the protein which is selected from the groupconsisting of HB9572 and HB9573.